1. Field of the Invention
The present invention relates to the field of molecular biology, immunogenetics, and nucleic acid chemistry. More specifically, it relates to methods and reagents for genotyping at the HLA Class I B locus. The invention therefore has applications in the field of medicine generally, and medical research and diagnostics specifically, the field of forensic science, and the field of molecular biology.
2. Description of Related Art
The major histocompatibility complex (MHC) includes a number of genes that encode glycoproteins that, together with the T cell receptor (TCR), are the key elements of specificity in the T cell response to foreign antigens. There are two structurally distinct, but related, families of MHC molecules that present antigens to two subsets of T cells: Class I MHC molecules present antigens to T cells that express the CD8 cell surface glycoprotein, and Class II MHC molecules present antigens to T cells that express the CD4 cell surface glycoprotein. See Bjorkman and Parham, 1990, Ann. Rev. Biochem. 59:253-288, incorporated herein by reference. For a general review of the HLA Class II genes and proteins, see Trowsdale et al., 1985, Immunol. Rev. 85:5; and Giles and Capra, 1985, Adv. Immunol. 37:1, both incorporated herein by reference.
The Class I gene products function as restriction elements in the presentation of mainly endogenous peptides to cytotoxic T lymphocytes and are the major barrier for allogeneic tissue transplantation. Accurate determination of allelic subtypes is essential for typing potential transplantation donors, where very precise HLA matching appears to be critical in minimizing risk of rejection or graft versus host disease.
Significant advances have been made in developing DNA based typing methods for determining the HLA Class H genotype of an individual. The polymerase chain reaction (PCR) is used to amplify a nucleic acid sequence encompassing polymorphic regions within the gene. Following amplification, the sequence variants present within the polymorphic regions are detected using sequence-specific oligonucleotide probes. For example, see Saiki et al., 1986, Nature, 324:163; Bugawan et al, 1988,J. Immunol. 141:4024-4030, Erlich and Bugawan, 1989, in: PCR Technology: Principles and Applications for DNA Amplification, ed. H. A. Erlich. Stockton Press, New York, N.Y.:193-208; Scharf et al., 1991, Human Immunol. 30:190-201; Erlich et al., 1991, Eur. J. Immunogen. 18:33-35, each incorporated herein by reference. Some of these DNA based typing methods are now commercially available for forensic, research and clinical use.
In contrast to the advances made in the field of HLA Class II DNA typing, little progress has been made in developing methods for HLA Class I DNA typing. One reason for this lack of progress is the complexity of the HLA Class I genes. The Class I includes the A, B, C, E, F, and G genes and the H, J, K, and L pseudogenes. The A, B, C, and E loci are known to each encode a large number of different alleles. The F and G loci are currently not believed to be polymorphic. The currently known differences are primarily in the second and third exons of these genes, although sequence variation in the fourth exon of these Class I genes is also known (see Zemmour and Parham, 1991, Immunogenetics 33:310-320, and Malissen et al., Feb. 1982, Proc. Natl. Acad. Sci USA79:893-897, both incorporated herein by reference).